highly-efficient-transfection-primary-macrophages-with-vitro

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

University of Cologne

Acid sphingomyelinase (ASMase) has been implemented in cellular signaling mainly because its reaction product, ceramide, has been assumed to be a mediator within signaling pathways. Our studies of three independent infection systems show that ASMase is required for phago-lysosomal fusion in macrophages infected with Listeria monocytogenes, for exocytosis of secretory lysosomes by lymphocytic choriomeningitis virus-specific cytotoxic T cells, and for generation of multinucleated giant cells in granuloma of mice infected with Mycobacterium avium. Because of its neutral lipid nature, ceramide is confined to the membranes of phagosomes and lysosomes or the extracellular leaflet of the plasma membrane. In light of the biochemical and biophysical properties of ceramide, we provide a model suggesting that ASMase regulates select vesicular fusion processes by modifying the steric conformation of cellular membranes.

Fusogenicity of membranes: the impact of acid sphingomyelinase on innate immune responses.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family. 

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo. 

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

The intracellular pathogen Listeria monocytogenes (L.m.) is targeted by the autophagic machinery, but the molecular mechanisms involved and consequences for anti-listerial immunity remain enigmatic. Here, we demonstrate that L.m. infection of macrophages in vivo exclusively evokes LC3-associated phagocytosis (LAP), but not canonical autophagy, and that targeting of L.m. by LAP is required for anti-listerial immunity. The pathway leading to LAP induction in response to L.m. infection emanates from the β integrin Mac-1 (CR3, integrin αβ), a receptor recognizing diverse microbial ligands. Interaction of L.m. with Mac-1 induces acid sphingomyelinase-mediated changes in membrane lipid composition that facilitate assembly and activation of the phagocyte NAPDH oxidase Nox2. Nox2-derived reactive oxygen species then trigger LC3 recruitment to L.m.-containing phagosomes by LAP. By promoting fusion of L.m.-containing phagosomes with lysosomes, LAP increases exposure of L.m. to bactericidal acid hydrolases, thereby enhancing anti-listerial activity of macrophages and immunity of mice.

The β Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes.

The macroautophagic/autophagic machinery cannot only target cell-endogenous components but also intracellular pathogenic bacteria such as Listeria monocytogenes. Listeria are targeted both by canonical autophagy and by a noncanonical form of autophagy referred to as LC3-associated phagocytosis (LAP). The molecular mechanisms involved and whether these processes contribute to anti-listerial immunity or rather provide Listeria with a replicative niche for persistent infection, however, remained unknown. Recently, using an in vivo mouse infection model, we have been able to demonstrate that Listeria in tissue macrophages are targeted exclusively by LAP. Furthermore, our data show that LAP is required for killing of Listeria by macrophages and thereby contributes to anti-listerial immunity of mice, whereas canonical autophagy is completely dispensable. Moreover, we have elucidated the molecular mechanisms that trigger LAP of Listeria and identified the integrin ITGAM-ITGB2/Mac-1/CR3/integrin αß as the receptor that initiates LAP in response to Listeria infection.

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes.

Corticosteroids are host-directed drugs with proven beneficial effect on survival of tuberculosis (TB) patients, but their precise mechanisms of action in this disease remain largely unknown. Here we show that corticosteroids such as dexamethasone inhibit necrotic cell death of cells infected with Mycobacterium tuberculosis (Mtb) by facilitating mitogen-activated protein kinase phosphatase 1 (MKP-1)-dependent dephosphorylation of p38 MAPK. Characterization of infected mixed lineage kinase domain-like (MLKL) and tumor necrosis factor receptor 1 (TNFR1) knockout cells show that the underlying mechanism is independent from TNFα-signaling and necroptosis. Our results link corticosteroid function and p38 MAPK inhibition to abrogation of necrotic cell death mediated by mitochondrial membrane permeability transition, and open new avenues for research on novel host-directed therapies (HDT).

Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition.

A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys and Cys, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

Phagocytes ingest, kill and degrade invading microbes in a process called phagocytosis. LC3-associated phagocytosis (LAP) combines the molecular machinery of phagocytosis with that of autophagy, the cellular pathway for ingestion of cytoplasmic components, resulting in the eponymous association of 'microtubule-associated proteins 1 A/1B light chain 3' (LC3) with the phagosomal membrane. The LC3-decorated phagosomes, or LAPosomes, show enhanced fusion with lysosomes resulting in enhanced killing and degradation of contained pathogens. Thus, LAP is a particularly microbicidal pathway. In this review, we discuss the molecular mechanisms involved in induction and execution of LAP and its crucial role in antimicrobial immunity against bacteria, fungi and parasites. As LAP has only recently been defined, we also point out the key open questions that remain to be answered.

Article	Year
Fusogenicity of membranes: the impact of acid sphingomyelinase on innate immune responses. Immunobiology\| PubMed ID: 18406376	2008
Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes. Nature immunology\| PubMed ID: 19525969	2009
Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature\| PubMed ID: 19641494	2009
Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 23842753	2013
Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes. European journal of immunology\| PubMed ID: 24272050	2014
Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors. Hepatology (Baltimore, Md.)\| PubMed ID: 28318036	2017
The β Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes. Cell host & microbe\| PubMed ID: 29544096	2018
LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes. Autophagy\| PubMed ID: 29923444	2018
Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition. Nature communications\| PubMed ID: 30737374	2019
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science signaling\| PubMed ID: 30755476	2019
LC3-associated phagocytosis - The highway to hell for phagocytosed microbes. Seminars in cell & developmental biology\| PubMed ID: 31029766	2019

Michael Schramm

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